Project Summary Early diagnosis of degenerative joint diseases like osteoarthritis (OA) is critical as there is only a narrow time- window during which therapeutic intervention can reverse disease progression. While computed tomography (CT) can diagnose changes in subchondral bone, it is not clinically viable for imaging joint soft tissues like cartilage which exhibit early degenerative changes associated with OA onset. CT may be developed for soft tissue imaging by using radio-opaque contrast agents injected into the joint, as long as they are safe and the time to produce optimal CT attenuation is short enough to be clinically viable. Contrast agents like ioxaglate are anionic and thus repelled by negatively charged cartilage that hinders their intra-tissue penetration and partitioning resulting in poor CT attenuation. This is further complicated by their short intra-tissue residence time owing to rapid clearance from joints, which necessitates high doses causing toxicity concerns. The high negative fixed charge density of cartilage, however, offers a unique opportunity to utilize electrostatic interactions to enhance intra-tissue transport, uptake, and retention of positively charged contrast agents. We previously showed, using avidin and polyarginine (R+20), that there exists an optimal size and charge range (diameter<10nm, charge +6 to +20) that can enable solutes to quickly penetrate through full thickness of cartilage following direct injection into joint, and have high uptake and retention. Based on these findings, this project will develop a new class of cationic contrast agents (CCA) that can rapidly penetrate through cartilage resulting in high uptake (30x higher than other contrast agents under development), before getting cleared out, thus producing sufficient CT attenuation at low doses and reducing the time between CCA administration and imaging to clinically viable levels. This will pave way for safe, effective CT imaging of negatively charged cartilage for early diagnosis of OA. In Aim 1, we will synthesize CCAs by conjugating avidin and R+20 to ioxaglate and then characterize their transport properties like diffusivity, partitioning factor, equilibrium uptake and binding constants in cartilage. We will also evaluate their dose dependent biological response using in vitro cartilage culture to estimate the maximum safe dose. In Aim 2, CCAs will be used to CT image normal and GAG depleted osteochondral plugs; optimal CCA concentration and incubation time to achieve depth dependent-spatial contrast enhanced CT attenuation in cartilage will be determined and CT data will be correlated with tissue's biochemical composition. In Aim 3, we will integrate our knowledge from Aims 1 and 2 to validate the efficacy of CCA in diagnosing cartilage degeneration associated with early stages of OA onset in vivo using anterior cruciate ligament transection (ACLT) model of post traumatic OA. This work has very significant implications on societal health as early OA diagnosis can enable timely therapeutic intervention while the disease is still reversible. CCAs developed here can be used for CT imaging of other negatively charged tissues like meniscus, intervertebral disc and eye.